Fig. S1. Southern blot analysis of the Medicago truncatula PI-like genes. MtPI and MtNGL9 are present in the Medicago genome as single copies.
Fig. S2. Molecular characterization of the Tnt1 insertions in the MtNGL9 locus. A. Schematic diagram of the genomic structure of the MtNGL9 gene. In NF14948 lines (mtngl9-1), Tnt1 was inserted 360 bp downstream of the ATG codon, at the second exon. In NF12316 lines (mtngl9-2). Tnt1 was inserted 183 bp downstream of the ATG codon, at the end of the first exon. B. Genotyping of mtngl9-1 mutant using NGL9-F/Tnt1-R (upper) and NGL9-291/NGL9612G (lower) primer pairs. Homozygous plants are indicated in circled numbers. C. Genotyping of mtngl9-2 mutant using NGL9-F/Tnt1-R (upper) and NGL9-F/NGL9-590G (lower) primer pairs. Homozygous plants are indicated in circled numbers. D. RT-PCR results show no detectable MtNGL9 expression in four homozygous mutant plants. MWM: molecular weight markers; WT: wild-type control.
Fig. S3. qRT-PCR expression analyses of loss-of-function plants. A. mRNA levels of the Bfunction genes in floral buds of mtpi-2. B. mRNA levels of the B-function genes in floral buds of mtngl9-1 and mtngl9-2. The height of the bars for a given gene indicates relative differences in expression levels in floral buds. The expression value of all B-function genes in WT was set to 1.00. Expression levels were plotted relative to this value.
Fig. S4. Analyses of 35S:MtPI and 35S:MtNGL9 plants. A. Transcript abundance of MtPI in 35S::MtPI plants. The expression value of MtPI in plant number 7 was set to 1.00. Expression levels were plotted relative to this value. Numbers indicate the transgenic line. Underlined number indicates plants classified as medium phenotype, and the remaining plants showed the strong phenotype. B. Transcript abundance of MtNGL9 in 35S::MtNGL9 plants. The expression value of MtPI in plant number 19 was set to 1.00. The number of the transgenic line is indicated. Underlined number indicates plants classified as weak phenotype, and the remaining plants showed the medium phenotype. C. Relative proportion of phenotypes observed in plants overexpressing MtPI (No of transgenic plants= 17) or MtNGL9 (No of transgenic plants= 33).
Fig. S5. RT-PCR expression analyses in pi-1/pi-1; 35S:MtPI and pi-1/pi-1; 35S:MtNGL9 complementation lines. A. Expression of MtPI in pi-1/pi-1; 35S:MtPI lines. B. Expression of MtNGL9 in pi-1/pi-1; 35S:MtNGL9 lines. C. Comparative expression level of MtPI and MtNGL9 in their respective complementation lines. Levels were quantified by measuring the intensity of individual bands using ImageJ densitometry software, and expressed relative to UBIQUITIN signal, as a measure of transcript relative abundance in the different samples. The numbers of the transgenic lines are indicated.
Fig. S6. Analyses of the pi-1 complementation by MtPI and MtNGL9. A. Genotyping of some individuals of the complementation lines by MtPI. B. Genotyping of some individuals of the complementation lines by MtNGL9. Genomic PCR product (729 bp) was digested by BseGI. pi-1/PI; 35::MtPI or pi-1/PI; 35S::MtNGL9 background resulting in three fragments: 729, 451 and 278 bp. Two fragments (278 and 451 bp) were obtained in the case of wild-type plants (PI/PI; 35S::MtPI or PI/PI; 35S::MtNGL9. BseGI does not cut the 729 bp PCR fragment in pi-1/pi-1; 35::MtPI or pi-1/pi-1; 35::MtNGL9 background (underlined numbers).
